High Clonal Diversity Mouse Model for Lineage Transcriptomic Epigenomic Single Cell Analysis

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Summary

In this article, a novel mouse model called the DARLIN was developed by the researchers to study tissue development and maintenance.This model, employing an inducible Cas9 system with terminal deoxynucleotidyl transferase and 30 CRISPR targets, surpasses previous lineage-tracing methods in barcode diversity and coverage. Remarkably, DARLIN tracks cell lineages in tissues with millions of cells, successfully identifying edited barcodes in about 70% of single cells examined. It was instrumental in uncovering the distinctive migration patterns of developing hematopoietic stem cells and their fate biases. Furthermore, DARLIN's innovative approach combines transcriptomic and epigenomic profiling at the single-cell level, revealing that cellular lineage memory correlates more with DNA methylation patterns than with gene expression or chromatin accessibility. This breakthrough paves the way for more detailed exploration of lineage dynamics and molecular characteristics across various tissues and conditions.

Fig.1 Graphical abstract¹.

Research Criteria

The study focuses on developing an improved mouse model named DARLIN for high-resolution lineage tracing. The model enhances the diversity and efficiency of lineage barcoding in single cells, enabling detailed profiling of lineage relationships and molecular states of cells in various tissues.

Sample Type

The single-cell experiments are conducted on samples from mice, specifically using hematopoietic stem cells (HSCs) and other blood progenitors. The samples are derived from various tissues like bone marrow, liver, and spleen.

Result—DARLIN Achieves Superior Coverage in Single Cell Lineage

The DARLIN mouse line represents a significant advancement in single-cell lineage tracing, outperforming previous methods like Cas9/CARLIN. This technique allows for the simultaneous profiling of lineage barcodes and transcriptomes in single cells, crucial for understanding cell states and lineage relationships in diverse populations. In a study involving DARLIN mice, a high percentage of cells from various tissues exhibited detectable, edited, and rare lineage barcodes, indicating efficient lineage tracing. Specifically, in blood cell progenitors from skull bone marrow, 81% of cells showed at least one target locus, with 63% having a rare allele. This efficiency was consistent across different tissues. In comparison, the DARLIN mouse line showed superior performance to the Cas9/CARLIN line, with a higher percentage of cells meeting all criteria for lineage tracing. This demonstrates DARLIN's enhanced capability in capturing a wide range of cell lineages, surpassing the diversity found in an entire adult animal.

Fig.2 Characterization of DARLIN mice¹.

Result—DNA Methylation Sustains Robust Clonal Recall Of Hematopoietic Stem Cells Through Time

The researchers then used Camellia-seq to analyze hematopoietic stem cells (HSCs) from adult bone marrow, focusing on clonal memory. They examined gene expression, chromatin accessibility, and DNA methylation. Results showed that while gene expression and chromatin accessibility indicated weak clonal memory, DNA methylation demonstrated strong clonal memory, with cells from the same clone having similar methylation patterns. The study identified 279 genomic regions with varying methylation across clones, unrelated to specific genes or functions. Further experiments across various samples confirmed DNA methylation as a more reliable indicator of clonal memory in HSCs than the other modalities, enhancing our understanding of stem cell identity and function over time.

Fig.3 HSCs' transcriptomic and epigenomic memory within each clone¹.

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Reference

1. Li, Li, et al. "A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells." Cell 186.23 (2023): 5183-5199.